The goal of the project is to understand the intramolecular interactions involved in autocatalytic RNA splicing, and the role of splicing in the regulatory biology of a simple organism, bacteriophage T4. Two new introns have been discovered in T4, and have localized near genes nrdB and nrdC. These introns will be characterized with respect to splicing intermediates, cofactors, and nucleotide sequence. Comparisons will be made with the characterized td intron of T4 and with other Class I introns of eucaryotes. Insertional mutagenesis of the introns and their flanking sequences, with a Beta-galactosidase fusion gene, will allow determination of the genes which contain the introns. Selection schemes have been devised to find mutations that block splicing, as well as mutations at a second site which reactivate it. Insertion of introns into the rIIB gene will provide a sensitive biological assay for splicing. The distribution of mutable sites in the introns will be analyzed to determine the interactions that contribute to the autocatalytic splicing mechanism. Also, a novel hypothesis, which suggests that the splicing reaction is coupled to a metabolic regulatory circuit, will be tested.